Animal cells are grown in culture to provide a number of pharmaceutical, diagnostic and veterinary products including human vaccines, lymphokines, hormones, monoclonal antibodies, other pharmaceutically active protein products, veterinary hormones and for research and development and diagnostic purposes.
The growth of animal cells requires a defined isotonic medium that contains salts, nutrients, lipid precursors, nucleic acid precursors, vitamins and amino acids that are formulated to mimic the medium that would normally bathe those cells in vivo. Examples in common use include Eagle's Minimal Essential Medium, Dulbecco's-modified Eagle's Minimal Essential Medium (DMEM), Medium 199, RPMI 1640 medium and Ham's F12 Medium. However, virtually no animal cells will grow in such a medium, but require the co-addition of serum. Fetal bovine serum is frequently used as it is more effective than serum obtained from postnatal animals and it contains only minimal concentrations of immunoglobulins which otherwise could have undesirable effects.
The supply of fetal bovine serum is limited by the number of pregnant cows slaughtered. It also has undesirable lot-to-lot variations and may include toxins. Particular concern surrounds its use for the eventual production of recombinant proteins and other pharmaceuticals for human use because the serum may also contain viruses that are harmful to humans and may be carried through a purification protocol that yields the desirable product. Principally for these reasons, extensive efforts have been directed towards the replacement of serum by pure ingredients. Examples of such ingredients are growth factors, hormones and cell attachment factors. Unfortunately, the requirements of each cell type being grown are different and are difficult to establish. Frequently it has not proved possible to achieve equivalent growth properties or equivalent yields of cell products with “serum-free” media as can be obtained with medium containing fetal bovine serum.
The limited availability of fetal bovine serum, its lot-to-lot variability, its resultant considerable cost as well as the deficiencies of “serum-free” media described above have prompted the investigation of other biological fluids as potential replacements in cell culture media. Some progress has been reported in the prior art with bovine milk and bovine colostrum as evidenced by the following selected reports: M. Klagsbrun: “Human milk stimulates DNA synthesis and cell proliferation in cultured fibroblasts” (Proc. Natl. Acad. Sci. USA 75, 5057, 1978); M. Klagsbrun & J. Neumann: “The serum-free growth of Balb/c 3T3 cells in medium supplemented with bovine colostrum” (J. Supramol. Struct. 11, 349, 1979).
The prior art also includes U.S. Pat. No. 4,440,860 to M. Klagsbrun which describes “compositions and methods for promoting cell growth featuring, in one aspect, cell culture media containing milk or colostrum and fibronectin; fibronectin is preferably pre-coated onto the culture substrate” and Japanese Patent JP 59166879 to Morinaga “A culture medium for cell incubation—containing milk or milk components”. Ultrafiltrates of milk whey have also been used to support the growth of cultured cells, as in O. Damerdji et al. “Utilization of whey fractions as a substitute for fetal calf serum in culture media” (Biotech. Tech. 2, 235, 1988).
The prior art also includes U.S. Pat. No. 5,077,276 “Growth Factor” to the applicants wherein the isolation of insulin-like growth factor IGF-I and des(1-3)IGF-I are described from bovine colostrum. A 25 kDa dimeric molecule, termed milk growth factor (MGF) has also been isolated from bovine milk by R. R. Burk et al. according to European Patent Application EP 0313515 and confirmed as transforming growth factor beta-2 (TGF-β2), (D. A. Cox and R. R. Burk, Eur. J. Biochem. 197, 353, 1991). The prior art also includes reports of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) in milk or colostrum (A. N. Corps. et al. J. Endocrinol. 112, 115, 1987).
Taken together, the prior art indicates that a range of cell growth stimulating factors are present in milk products, although a method for their isolation as a mixture to stimulate the growth of cultured cells that are separated from the major proteins in milk has not been described. Despite this progress a successful alternative to fetal bovine serum is yet to be located.
It is accordingly an object of the present invention to overcome, or at least alleviate one or more of the difficulties or deficiencies related to the prior art.